Rapid Detection of Bacteria using Mass Spectrometric Analysis

ABSTRACT

Methods for the detection or diagnosis of a bacterial infection or colonisation utilising mass spectrometric analysis are provided. The methods involve short-term enrichment of samples followed by mass spectrometric analysis of biomarker profiles. Also provided are methods for preparing short-term enrichment cultures.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/147,951, having a filing date of Aug. 4, 2011, which applicationclaims priority under 35 U.S.C. §371 to International Application No.PCT/GB2010/000219, filed Feb. 5, 2010, which application claims priorityto Great Britain Application No. 0902033.0, filed Feb. 6, 2009, thedisclosure of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to a method for the detection or diagnosis of abacterial infection or colonisation. More specifically, it relates todetection of the presence or absence of particular bacteria using massspectrometric analysis, in particular MALDI-TOF mass spectrometricanalysis.

BACKGROUND

For effective management of bacterial infection, early diagnosis of thepresence of an infection/colonisation and swift treatment is essential.A particular problem in clinical environments such as hospitals isdetecting, isolating, and treating patients and health workers carrying‘superbugs’ such as MRSA (methicillin-resistant Staphylococcus aureus)before they come into contact with vulnerable patients. The prevalenceof infections caused by MRSA has been increasing for several years inmany countries around the world.

Reports from the UK National Audit Office state that at any one time, 9per cent of NHS hospital patients are suffering from an infection suchas MRSA, acquired whilst in surgery or as an inpatient on the hospitalwards. These ‘nosocomial’ infections affect 100,000 people annually,costing the National Health Service £1 billion (

1.5 bn), and causing up to 5,000 deaths. The US Centre for DiseaseControl and Prevention estimates that between 60,000 and 80,000Americans die each year from nosocomial infections, and the cause in themajority of cases is S. aureus.

Some strains of MRSA are particularly successful at spreading betweenpatients and may also spread between hospitals, for example whencolonised patients or staff move from one hospital to another. Thesestrains are known as epidemic MRSA (EMRSA). During the 1990s there was amarked increase in infections caused by MRSA in hospitals in the UK dueto the emergence and spread of two particular stains of EMRSA known asEMRSA-15 and EMRSA-16. In 2002, a new epidemic strain, EMRSA-17, wasdescribed in the UK. This strain is more resistant than any previous UKEMRSA strains.

Older methods of screening are too slow to effectively prevent thespread of hospital-acquired infections and can be very expensive andlabour-intensive. Some existing methods are also inaccurate and maysuffer from problems with specificity and sensitivity. Laboratoryscreening for MRSA is therefore a complex balance between speed ofresult, sensitivity, specificity and cost.

Standard methods for diagnosis of MRSA involve overnight culturing ofthe bacteria followed by visual identification and verification of MRSAcolonies. Agar plate-based methods are the most common, using eitherselective or non-selective media. Broths or slopes (which may also beselective or non-selective) can also be used. These methods have beenpractised for many years, and are generally quite specific andsensitive. However, they can provide answers only after 3-5 days, whichis of little use in a hospital setting, from the perspective ofinfection control.

As a result, newer methods, in particular PCR based methods, have beendeveloped with the aim of providing results more rapidly. PCR resultscan generally be obtained in one working day, compared to three workingdays for culture methods, although this is still too long if the spreadof the bacteria is to be effectively controlled.

The ‘first generation’ of PCR-based methods (e.g. References 1-4) relyon the presence of a combination of the methicillin resistance gene,mecA, with one or more other genes specific to S. aureus (e.g. femA ornuc). These methods have major drawbacks in the form of false positiverates, costs, and lack of automation, and are mostly only suitable foruse with pure cultures, not screening of clinical swabs.

The more recently developed generation of PCR-based methods (e.g.References 5-8) have had some success. For example, the IDI-MRSA PCRtest, in which a single genetic sequence is detected specific to MRSA,is now approved for use in nasal swabs for colonisation. These methodsstill suffer from problems, however. Using nasal swabs only, they willonly catch 50% of the patients who are colonised, and none of those withbacteraemia.

Other methods which have been developed include the latex agglutinationmethod (Reference 9). The most commonly used commercial test relies onthe presence of Protein A and PBP2A which, combined together, pointtoward MRSA. However, this test has sensitivity and specificityproblems.

There is a need for a new method for the rapid diagnosis of bacterialinfections, such as MRSA, which overcomes the sensitivity andspecificity problems of currently available methods, and can beperformed quickly, easily and at low cost.

SUMMARY OF THE INVENTION

The present invention provides detection methods for bacterialinfections or colonisations, which utilise short-term enrichmentfollowed by mass spectrometric analysis of the bacterial biomarkerprofile.

Accordingly, in one aspect, the invention provides a method fordetection of the presence or absence of particular bacteria in a sample,the method comprising:

-   -   i) enriching the sample by culturing the bacteria present in the        sample, for a period of less than six hours;    -   ii) analysing the enriched sample by mass spectrometry, to        obtain biomarker profile data for the sample;    -   iii) comparing the sample biomarker profile data with reference        data, to determine the presence or absence of said particular        bacteria in the sample; wherein the reference data is obtained        from a reference sample of said particular bacteria,    -   wherein the reference sample has been cultured for less than six        hours.

In a related aspect, the invention provides a method for detection ofthe presence or absence of particular bacteria in a sample, the methodcomprising:

-   -   (i) enriching the sample by culturing until a detectable        quantity of pre-modification biomarkers is produced in the        sample;    -   (ii) analysing the enriched sample by mass spectrometry, to        obtain biomarker profile data for the sample;    -   (iii) comparing the sample biomarker profile data with reference        data, to determine the presence or absence of said particular        bacteria in the sample;    -   wherein the reference data relates to pre-modification        biomarkers produced by a reference sample of said particular        bacteria.

In another related aspect, the invention provides a method for detectionof the presence or absence of particular bacteria in a sample, themethod comprising:

-   -   (i) enriching the sample by culturing the bacteria present in        the sample;    -   (ii) analysing the enriched sample by mass spectrometry, to        obtain biomarker profile data for the sample, wherein the        biomarker profile data comprises peaks corresponding to        pre-modification biomarkers produced by the bacteria in the        sample;    -   (iii) comparing the sample biomarker profile data with reference        data, to determine the presence or absence of said particular        bacteria in the sample;    -   wherein the reference data relates to pre-modification        biomarkers produced by a reference sample of said particular        bacteria.

A further related aspect of the invention provides a method fordetection of the presence or absence of particular bacteria in a sample,the method comprising:

-   -   (i) enriching the sample by culturing the bacteria present in        the sample;    -   (ii) analysing the enriched sample by mass spectrometry, to        obtain biomarker profile data for the sample, wherein the        biomarker profile data comprises peaks in the region below 1500        Da;    -   (iii) comparing the sample biomarker profile data with reference        data, to determine the presence or absence of said particular        bacteria in the sample;    -   wherein the reference data relates to peaks in the region below        1500 Da produced by a reference sample of said particular        bacteria.

The methods of the invention are for detecting the presence or absenceof particular bacteria in a sample i.e. for detecting a particularbacterial infection or colonisation of interest. The bacterialinfection/colonisation to be detected is typically pre-determined andmay comprise a particular bacterial strain, sub-species, species orgenus. The reference data to which the sample mass spectrometric profileis compared in the methods of the invention is chosen according to theparticular strain, species or genus of bacteria to be detected, asfurther described below. Any type of bacteria for which a short-termenrichment biomarker profile can be established, as described herein,may be detectable using the present methods.

Examples of bacteria which may be detected include Staphylococci (suchas S. aureus, S. afermentans, S. auricularis, S. capitis, S. caprae, S.cohnii, S. epidermidis, S. felis, S. haemolyticus, S. hominis, S.intermedius, S. lugdunensis, S. pettenkoferi, S. saprophyticus, S.schleiferi, S. simulans, S. vitulus, S. warneri, S. xylosus; preferablyStaphylococcus aureus), Clostridium species (such as C. difficile, C.botulinum, C. perfringens, C. tetani; especially C. difficile),Escherichia coli, Campylobacter (such as C. jejuni, C. coli and C.fetus), Salmonella, Pseudomonas, Shigella, Neisseria, Klebsiella,Vibrio, Legionella, H influenzae, H pylori, Bacillus, Listeria as wellas tuberculosis and leprosy.

In some preferred embodiments, the methods of the invention are used todetect the presence of Gram-positive bacteria. More preferably, thebacterium is Staphylococcus, such as Staphylococcus aureus. Inparticularly preferred embodiments, the bacterium ismethicillin-resistant Staphylococcus aureus (MRSA). MRSA refers tocertain strains of Staphylococcus aureus which are resistant tomethicillin and similar antibiotics. In particular, it is useful todetect certain strains known as epidemic MRSA (EMRSA) for exampleEMRSA-15, EMRSA-16 and EMRSA-17.

The sample analysed in the methods of the invention may be any samplesuspected of containing a particular bacterial infection orcolonisation. Samples may be taken from humans or non-human animals,particularly mammals, or from a source which is not directly a livinganimal for example from food or from a building or an industrial plant,such from a hospital structure (e g hospital floors or furnishings), awater-containing system in a building (e g water supply, heating,cooling or air-conditioning system) or a food-processing plant. Inpreferred embodiments, the sample to be tested is a clinical sampletaken from a human patient. It may be in the form of a blood sample, atissue sample, a urine sample, a fecal sample, a gastric sample, asaliva sample, a cerebrospinal fluid sample or a swab, for example anasal swab.

In some preferred embodiments, the sample is taken from a wound site, anulcer, or a human screening site (e.g. nose, throat, groin, perineum,axilla).

Advantageously, the method may detect very small concentrations ofbacteria. This may enable detection of colonisation as well asinfection. Colonisation is defined as the presence of proliferatingbacteria without a host response and usually involves lowerconcentrations of bacteria thus making it harder to detect (see e.g.References 16 and 17).

Advantageously, diagnosis may be possible starting from a samplecontaining a wound concentration of bacteria.

The method may allow detection of concentrations as low as 1 cfu/ml(colony forming units/ml). 1 cfu/ml is equal to 1 bacterium that canform a colony by binary fission. This means that there is 1 viablecolony forming organism in that ml of sample. Microbiology baseddetection uses cfu/ml as the standard measurement of microbialconcentration.

In some embodiments, the sample, before enrichment, may contain bacteriaconcentration of about 10⁹ cfu/ml or below; about 10⁸ cfu/ml or below;about 10⁷ cfu/or below; about 10⁶ cfu/ml or below; about 10⁵ cfu/ml orbelow; about 10⁴ cfu/ml or below; about 10³ cfu/ml or below; about 10²cfu/ml or below; about 10 cfu/ml or below; or about 1 cfu/ml or below.

In some embodiments the sample, before enrichment, contains bacteria ata concentration of about 1 cfu/ml to about 10⁹ cfu/ml; about 10 cfu/mlto about 10⁹ cfu/ml; about 10² cfu/ml to about 10⁹ cfu/ml; about 10³cfu/ml to about 10⁹ cfu/ml; about 10⁴ cfu/ml to about 10⁹ cfu/ml; orabout 10⁵ cfu/ml to about 10⁹ cfu/ml.

The sample is prepared for analysis by a short-term enrichment process.The number of bacteria and hence the amount of bacterially producedproteins in the sample are increased in this process. Enrichment of thetest sample is carried out by culturing the sample. Culturing ispreferably carried out for a pre-determined time period, which ispreferably for less than 6 hours, for 4 hours or less, for 2 hours orless, for 1 hour or less, or for 30 minutes or less. For practicalreasons, a preferred minimum time period may be 5 minutes, 15 minutes,30 minutes, 1 hour, or 90 minutes.

In some embodiments of the invention, the biomarker profile data for thesample comprises mass spectrometric peaks with m/z values in the regionbelow 1500. This typically corresponds to molecules having molecularmass in the region below 1500 Da. The biomarker profile data for thesample may therefore be said to comprise mass spectrometric peaks in theregion below 1500 Da. These peaks may be present in addition to peakswhich are also present in a 24 h-culture biomarker profile for thebacteria.

The profile data obtained from analysis of the short-term enrichedculture preferably includes data relating to pre-modification biomarkersproduced by said particular bacteria. The mass spectrometric profiletherefore comprises peaks corresponding to said pre-modificationbiomarkers produced by the bacteria in the sample.

In some embodiments, the pre-modification biomarkers may comprisepre-modification proteins and/or peptidoglycans produced by saidparticular bacteria. The pre-modification biomarkers may comprisepre-PTM (post-translational modification) proteins. In certainembodiments, the pre-modification proteins may comprise unglycated cellwall peptides. In some preferred cases, these pre-modification proteinscomprise proteins or peptidoglycans with a molecular mass below 1500 Da.

A wide variety of culture media and culture conditions are known tothose skilled in the art, including plates (e.g. Agar plates), slopes,slants or broths. The exact nature of the culture media which may beused in the short-term enrichment culture step of the methods of theinvention may be varied, to optimise enrichment and to ensurereproducibility of the biomarker profile. In some embodiments of theinvention, the culturing is carried out in a broth, such as a BrainHeart Infusion (BHI) broth, a Mueller-Hinton Broth, an Anaerobic brothor a Nutrient Broth. In preferred embodiments a Brain Heart InfusionBroth is used. Optionally, a broth may contain additives such asantibiotics.

The present inventor has developed a novel culturing and enrichmentmethod for bacterial samples, which is particularly advantageous whenused in conjunction with the MS analysis method of the presentinvention. Accordingly, a further aspect of the present inventionprovides a method of obtaining an enriched bacteria-containing sample,including the steps of:

-   -   inoculating a bacteria-containing sample into a broth;    -   culturing the sample-containing broth for a period of less than        6 hours;    -   separating the enriched sample from the sample-containing broth        by centrifugation.    -   Culturing the sample preferably comprises incubation in a water        bath.

An enriched sample may need to be prepared or processed prior toanalysis, using known methods. For example, bacterial colonies may beremoved, separated or isolated from the culture medium. If the culturingis carried out in a broth, separation of the bacterial colonies mayinvolve centrifugation of the broth and removal of the supernatantliquid, such as in the enrichment process discussed above.

Preferably the centrifugation step in the enrichment process comprises acentrifugation and pooling protocol, to improve the separation of thesolid and liquid components of the sample-containing broth. Thisprotocol comprises:

-   -   dividing the sample-containing broth into a plurality of        aliquots;    -   centrifuging the aliquots;    -   discarding the resultant supernatants;    -   re-suspending the pellets in water;    -   pooling the resultant pellets; and    -   centrifuging a suspension of the pooled pellets, to separate the        pellet containing the enriched sample.

Some examples of broths suitable for culturing bacteria are discussedabove. Preferably the broth is prepared by reconstitution of a powderedmicrobiological culture material. More preferably, preparation of thebroth involves reconstituting the broth in water, autoclaving thereconstituted solution at a temperature between 80 and 150° C.,preferably from 110-125° C., most preferably about 120° C. or 121° C.,for 10-30 minutes preferably about 15 minutes; leaving the solution tocool to a pre-determined temperature in the autoclave; removing thesolution from the autoclave once this pre-determined temperature isreached and then allowing the solution to continue cooling at roomtemperature. The pre-determined temperature may vary depending on thenature of the broth, but is typically from 50-100° C., preferably from60-90° C., more preferably around 80° C.

The incubation of the sample for culturing is preferably performed in awater bath i.e. by ‘wet incubation’. The incubation is carried out at apre-determined temperature for a pre-determined length of time.Advantageously this method allows culturing to be carried out for ashort time period (less than 6 hours). More preferably, the sample isincubated for less than 4 hours, less than 2 hours or less than 1 hour.In some preferred embodiments, a culture time (incubation time) of from30 minutes to 2 hours is used. The optimal temperature for incubationmay vary depending on the organism to be cultured. In some preferredembodiments the temperature is from 25 to 60° C., more preferably from30 to 40° C., or from 30 to 37° C. In some embodiments culturing iscarried out at about 37° C. The temperature for incubation may becontrolled by thermostatic control of the temperature of the water bath.

After enrichment as described above, analysis of the sample ispreferably carried out using mass spectrometry, in accordance with thedetection methods of the invention.

In preferred embodiments, the analysis of the sample after short-termenrichment is performed using MALDI-TOF mass spectrometry, mostpreferably intact cell MALDI-TOF (ICM) analysis. In these embodimentspreparation of the sample may involve applying bacterial isolates to aslide and layering with a suitable matrix material, as is known in theart.

The presence or absence of the particular bacteria of interest isdetermined in the methods of the invention by comparison of the profiledata obtained from the mass spectrometric analysis of the short-termenriched sample with mass spectrometric reference data. The referencedata is obtained from a reference sample of the bacteria of interest,which has been cultured and analysed, for example under analogousconditions to those which will be used on the test samples. In someembodiments the reference data comprises a reference mass-spectrometricprofile obtained from a reference sample of a pre-determined bacterialinfection/colonisation (i.e. the bacterial strain or species to bedetected), which has been cultured for less than six hours. In someembodiments the reference data relates to pre-modification biomarkers,for example pre-modification proteins and/or peptidoglycans, produced bythe particular bacteria. In some methods, the reference data relates topeaks in the mass spectrum of a short-term enriched reference sample ofthe bacterial species to be identified, in the region below about 1500Da.

Reference data for a particular bacteria can be obtained from areference sample of said particular bacteria. Any of the enrichment andanalytical methods described herein for the test sample may equally beapplied to a reference sample. For example, a reference sample may beobtained from a pure culture of the bacteria of interest, which may besampled and subjected to short-term enrichment, as described herein.

Apparatus for performing the methods of the invention is also provided.Preferably the apparatus comprises a mass spectrometer and a dataprocessor, the processor being programmed with said reference data foridentifying one or more bacterial infections. The mass spectrometer ispreferably a MALDI-TOF mass spectrometer.

In some preferred embodiments, the apparatus comprises an automatedsystem for culturing a sample for subsequent analysis in the massspectrometer. The automated system preferably comprises: an incubatorfor heating the sample during culturing; a temperature control devicefor controlling the temperature of the incubator; and a centrifuge forseparation of the cultured sample from the culture medium.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the process of serial dilution of a culture in BHIbroth, to emulate bacterial concentrations frequently found at a woundsite.

FIG. 2 illustrates the division of a short-term enriched sample into tenaliquots. Ten aliquots of one milliliter are created from one sample often millilitres.

FIG. 3a-e illustrates the preparation of a MALDI-TOF slide: (a) A KratosKompact twenty well MALDI-TOF slide is used. Each well is 2 mm indiameter. (b) The slide was layered with the bacterial isolates. (c) Thebacterial layered slide was allowed to dry. (d) This slide was layeredwith matrix (CMBT) in two serial applications . (e) The matrix layeredslide was allowed to dry, for up to 45 minutes, before it was introducedinto the MALDI-TOF for analysis.

FIG. 4 demonstrates the working of a MALDI-TOF spectrometer.

FIG. 5 shows the result of a cluster analysis for sequence dissimilarityon five replicates of one isolate.

FIG. 6 shows the provisional diagnostic profiles for four bacteria (fromtop): Staphylococcus aureus (MSSA), Endemic Methicillin ResistantStaphylococcus aureus 15(EMRSA15), Endemic Methicillin ResistantStaphylococcus aureus 16(EMRSA15) and Coagulase negative Staphylococci(CNS), after short term enrichment at 4 hours in the mass range of 1.5-2kDa.

FIG. 7 demonstrates that the peak profile is maintained across thespectrum of short term enrichment. The profiles at the bottom of thefigure are obtained at two hours, those in the middle at four hours andthose at the top at six hours of the same isolate of S. aureus.

FIG. 8 compares overnight growth on an agar plate (top) to profilesobtained after enrichment at 4 hours (bottom) of the same bacteriumEndemic Methicillin Resistant Staphylococcus aureus 15(EMRSA15) in themass range of 1-4 kDa.

DETAILED DESCRIPTION OF THE INVENTION

The present inventor has found that biomarker profiles of bacterialspecies obtained after short-term enrichment culture are of use in thedetection and diagnosis of infection or colonisation. These biomarkerprofiles can readily be examined by mass spectrometric analysis.

The biomarker profile data obtained and used in the methods of theinvention may comprise proteomic profile data.

The term ‘proteome’ is commonly used to describe the entire complementof proteins produced by an organism or system, including modificationsmade to particular proteins. The proteome of an organism will vary withtime, and will also depend on the various stresses that a cell ororganism undergoes. As used herein, the term ‘proteomic profile’ refersto information about the protein content of a sample, as characterisedby peaks in its mass spectrum corresponding to the proteins,glycoproteins, glycopeptides, peptidoglycans, and other species makingup the proteome of the bacteria present in the sample. ‘Proteomicprofile data’ is data relating to all or part of the proteomic profileof the sample, for example, mass spectrometric data, such as m/z valuesof peaks in the mass spectrum.

It is also possible that substances which are not proteins arerepresented in the mass spectrum, for example carbohydrates orlipo-polysaccharides, as well as glycoproteins, glycopeptides,peptidoglycans, and other species as mentioned above. For the sake ofsimplicity, however, the terms “proteomic profile” and “biomarkerprofile” may be used interchangeably herein, said profiles beingrepresented by the peaks in the mass spectra of the bacteria.

In some preferred embodiments, the biomarker profile data comprises thevalues of peaks in the mass spectrum relating to the ‘young’ or ‘early’biomarker profile of the sample i.e. to proteins and related moleculespresent in the sample after a short-term enrichment culture, as furtherexplained herein.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry isa well known technique which uses ‘soft’ ionization, so allowing theanalysis of biomolecules (biopolymers such as proteins, peptides,peptidoglycans, sugars etc) which tend to fragment under conventionalionization conditions. Ionization is triggered by a laser beam (normallya nitrogen laser). A matrix is used to protect the biomolecule frombeing destroyed by the beam, and to facilitate vaporization andionization. The matrix consists of crystallized molecules.

The identity of suitable matrix compounds is determined to some extentby trial and error and depends on the sample to be analysed, as is knownin the art. However, conventional matrix materials are generally of alow enough molecular weight to allow facile vaporization but have a lowenough vapour pressure not to evaporate during sample preparation orwhile standing in the spectrometer. They are also generally acidicmolecules, thus acting as a proton source to encourage ionization of theanalyte, and have strong UV absorption, so that they rapidly andefficiently absorb the laser irradiation. They may be functionalizedwith polar groups, allowing their use in aqueous solutions.

Commonly used matrices are 3,5-dimethoxy-4-hydroxycinnamic acid(sinapinic acid), α-cyano-4-hydroxycinnamic acid (alpha-cyano oralpha-matrix) and 2,5-dihydroxybenzoic acid (DHB), picolinic acid (PA)and 3-hydroxy picolinic acid (HPA). For applications with MRSA, apreferred matrix is 5-chloro-2-mercaptobenzothiazole (CM BT).

In a standard method of sample preparation, a solution of one of thesematrix molecules is made, often in a mixture of highly purified waterand an organic solvent (normally acetonitrile or ethanol). The matrixsolution is then mixed with the analyte (i.e. the sample) and thissolution is spotted onto a MALDI plate. The solvents vaporize, leavingthe matrix and analyte co-crystallized in a MALDI ‘spot’, on which thelaser is fired to produce the ions which are then detected and analysed.The mass-to-charge ratio of the particles can be calculated based onbehaviour of the ions as they pass through the electric and magneticfields generated by the MS instrument.

Typically, a time-of-flight (TOF) analyzer uses an electric field toaccelerate ions through the same potential, and then measures the timethey take to reach the detector. If the particles all have the samecharge, the kinetic energies will be identical, and their velocitieswill depend only on their masses. Lighter ions will reach the detectorfirst.

Proteomic profiles of bacterial organisms, obtained by MALDI-TOF-MSanalysis, have previously been obtained and used for typing anddiscrimination of bacterial organisms, including Staphylococci(References 10-14). In general, bacterial proteomic profiles foranalysis have been obtained only after a long-term (usually overnight)culturing process. For example, a technique for acquiring a massspectrometric ‘fingerprint’ of MRSA was developed (Reference 11) inwhich the optimum fingerprint was achieved after 18-24 hours incubation,although a 6 hour incubation period was also tested.

The mass spectrometry-based techniques currently used in bacterialtyping are not easily applicable to direct analysis of clinical samples.Typing work is primarily aimed at identifying bacteria in an epidemicsetting, not at a clinical diagnosis. Therefore, the timescale fortyping is not usually critical—up to 24 hours can be taken to perform atyping analysis, without this making a huge difference to the outcomefor patients. Conversely, the time taken for detection and diagnosis ina clinical setting is more important since it can make a difference tothe outcome for patients.

The proteomic profile produced in the typing work, after a 24 hourculture of MRSA, comprises characteristic ‘biomarker’ peaks, which arepredominantly in the higher molecular weight region of the spectrum. Theproteins responsible for these peaks have not been identified but it isthought that they could represent mature (i.e. after post-translationalmodification) bacterial cell wall proteins. The biomarker peaks used inthe prior art cannot be reliably detected from cultures after only twohours.

GB 2 438 066 A (Bruker Daltonik GmbH) describes a method of measuringthe resistance of microbes to specific antibiotics, which involvesincubating said microbes in an antibiotic-containing medium andsubsequent analysis by mass spectrometry. Incubation times of around twohours are suggested. In this time the effect of the antibiotic in themedium (if any) on the growth of the bacteria can be assessed. However,there is no suggestion that a stable and reproducible diagnosticbiomarker profile can be obtained, starting from a clinical sample, inthis timeframe. Rather, this method looks at changes in the massspectrometric profile (typically in the 5,000-20,000 Da range) caused bythe presence of the antibiotic. The mass spectra from these incubatedsamples is compared to spectral libraries, which typically comprisereference data from ‘mature’ colonies e.g. 24 hour cultures.

The present inventor has now surprisingly found that diagnosticallyuseful biomarker profile data can be reproducibly obtained aftershort-term enrichment (i.e. after only a few hours culturing). This hasopened up, for the first time, a real possibility for providing anautomated, relatively foolproof, method for the rapid diagnosis ofinfections using mass spectrometry, which will be usable in a real-lifeclinical setting such as a public hospital.

The peak profiles obtained after short-term culture are significantlydifferent to those obtained at 24 hours growth, containing a much largernumber of low molecular weight (e.g. m/z below 1500; molecular massbelow 1500 Da) peaks (e.g. FIG. 8). These low molecular weight peaks arenot present in the proteomic profiles obtained after long-term(overnight) culture. It is thought that the lower molecular weight areaof the mass spectrum has never previously been looked at or studied.

Without wishing to be bound by theory, it is thought that the lowmolecular weight peaks, found in the short term enriched biomarkerprofile, represent bacterial proteins or peptidoglycans, probably fromthe bacterial cell wall, in an unmodified state—i.e. beforepost-translational modification. These peaks represent unique biomarkerswhich are useful for fast detection and identification of bacteria inclinical samples. In some cases, further unique biomarker peaks, in thehigher mass range of the spectrum, corresponding to pre-modificationbiomarkers of higher molecular weight, may also be present in theshort-term enriched biomarker profile.

Post-translational modification (PTM) is the chemical modification of aprotein after its translation and is one of the later steps in proteinbiosynthesis for many proteins. After translation, in which proteins areproduced in the cell by decoding mRNA produced from transcription of thegenome, post-translational modification then extends the range offunctions of the protein by attaching to it other biochemical functionalgroups such as acetate, phosphate, various lipids and carbohydrates, bychanging the chemical nature of an amino acid (e.g. citrullination) orby making structural changes, like the formation of disulfide bridges.In the case of bacterial cell wall proteins, an importantpost-translational modification is glycation of the peptides, i.e. theaddition of a sugar molecule to the protein.

It is proposed that the proteins present in a ‘young’ bacterial sample,i.e. a sample which has been subjected to only short-term enrichment,may contain a higher proportion of unmodified peptides(‘pre-modification’) i.e. peptides which have been produced bytranslation but have not yet been modified to their final form.

Similarly, it is proposed that other biomarkers may be different in‘young’ samples because a biomarker species has been produced by thebacteria but has not yet been modified to its final form, i.e. the formfound in mature bacteria. A sample which has been subjected to onlyshort-term enrichment may therefore contain a higher proportion ofunmodified biomarkers (‘pre-modification’) i.e. biomarkers which havebeen produced but not yet been modified to their final form.

Given the transient nature of these unmodified species, it would nothave been expected that a biomarker profile obtained after only a fewhours would be reproducible and characteristic of the presence of aparticular type of bacteria.

The present inventor has shown, however, that samples containing a lowconcentration of bacteria (e.g. 10⁵ cfu/ml—typical of clinical samples,such as from a wound site) can be cultured for a short time period, forexample for 2 hours or less, and that the resultant ‘short-termenriched’ sample produces a useful diagnostic biomarker profile when thesample is subjected to proteomic analysis, for example an intact cellMALDI-TOF (ICM) analysis. Even shorter culture times and smaller numbersof organisms are also envisaged.

As used herein the term ‘enrichment’ refers to a process of increasingthe number of bacteria present in the sample i.e. by culturing in asuitable medium. ‘Short-term enrichment’ implies that the culturing iscarried out for a relatively short time period, as compared toconventional bacterial culturing, which generally takes place overnight.Typically, short-term enrichment means culturing the sample for lessthan 6 hours. Shorter culture times are more preferred, for example 4hours or less, 2 hours or less, 1 hour or less, 30 minutes or less. Atypical culture time is from 30 minutes to 2 hours. The ‘culture time’is measured from the moment that the bacteria-containing (or suspectedbacteria-containing) sample is inoculated into a culture medium. Thesample may be heated (incubated) at a pre-determined culture temperaturefor all or part of the culture time. In the methods of the invention,culturing ends when the bacteria-containing portion of the enrichedsample is separated from the culture medium.

As shown in more detail in the Examples, below, a ‘young’ (short-termenriched) biomarker profile for several Staphylococcus strains hasalready been established (FIG. 6) and has been shown to be maintainedwith 100% reproducibility, in the mass range of 1-4 kDa, across thespectrum of short-term enrichment (2 to 6 hours enrichment).

Results were obtained using optimised short-term enrichment cultureconditions consisting of a custom culture broth, prepared from readilyavailable starting materials, as set out in the Examples, below. Thereare a wide variety of broth based media available, in addition to theBHI (Brain Heart Infusion) broth used in this example. Other broths suchas Mueller-Hinton Broth, Anaerobic broth and Nutrient Broth may also beused. Broths enriched with antibiotics may be useful, to ensureselective growth of the bacteria of interest (the bacterial infection tobe detected), thus simplifying the analysis. The best conditions forperforming the culture will depend on the organism to be detected.

Conventional broths are commercially available and can be obtained readyprepared in liquid form. Alternatively, the microbiological culturematerials making up the broth can be obtained in powder form andre-constituted, as is known in the art. The present inventor has foundthat better results are achieved if the broth is carefully prepared,from powdered starting materials, using a new method. Conventionally,the powdered ingredients are reconstituted in distilled water and thensterilised by autoclaving. Autoclaving is carried out at hightemperature, for example at around 120° C., although this may varydepending on the broth being prepared. The broth is then left to cool toroom temperature before use. It is usual to leave the reconstitutedsolution in the autoclave until it has reached room temperature and iscool enough to be handled, which typically takes around two to threehours.

However, the present inventor has found that if the solution is removedfrom the autoclave at a higher temperature, a better broth, which givesbetter results for short-term culturing, is obtained. Without wishing tobe bound by theory, it is thought that leaving the broth in the highertemperatures within the autoclave results in the meat in the broth beingbroken down into smaller molecules, which are difficult for the bacteriato consume, and that there may be some other inhibiting factor which isreleased which decreases growth in the first two to four hours ofculturing.

The culture medium, for example as prepared above, is inoculated withthe sample to be tested and the sample is cultured, to increase theamount of bacteria present in the sample. Conventional methods for theculturing of bacteria, particularly MRSA, tend to use a dry incubationi.e. incubation in a dry environment such as a dry incubator. Dryincubation relies on convection as a means of heating the medium andhence is non-uniform. Colder areas on the plate/broth may be presentwhich will affect the growth of bacteria in those areas. The use of ‘wetincubation’, i.e. incubation in a water bath or similar environment, maybe messier, but it is thought that it ensures uniform heating of theculture media, which helps the bacteria to grow more quickly. Thetemperature at which the water bath is set will vary depending on theculture conditions to be used. These can be optimised for each organismto be detected. Typically, temperatures close to human body temperature(i.e. 37° C.) are used, although MRSA can be cultured temperatures of30-60° C.

The bacteria content in the cultured sample is analysed after culturingto obtain its biomarker profile. In conventional culture methods, usingAgar based media, visualisation of the bacterial colonies growing on theplate or slope is needed, in order for these colonies to be picked outfrom the medium and analysed. In the preferred method of the presentinvention, a broth culture medium is used, and a new method ofseparating out the bacteria-containing material from the medium bycentrifugation has been developed. For example, the cultured sample issplit into several aliquots (for example, a 10 ml sample can be splitinto ten 1 ml aliquots (see FIG. 2) and centrifuged at 13,500 rpm forabout three minutes in a microcentrifuge. The supernatant can bediscarded and the pellets are then re-suspended in about 10 μl water.The resuspended aliquots are pooled together. The pooled sample is thenre-centrifuged (e.g. at 13,500 rpm for a further three minutes) and theresulting pellet is used for mass spectrometric analysis. As will beclear to the person skilled in the art, a short-term enriched sampleobtained in this way could equally be subjected to other types ofanalysis, as well as mass spectrometry. For example, applying thePCR-based methods, or latex agglutination methods, discussed earlier, toa culture prepared in only two hours, according to the present method,could potentially improve the speed and sensitivity of these methods ofdiagnosis.

In some embodiments, the culture medium is preferably free ofantibiotics i.e. culturing is performed in a non-selective manner. Thepresence of antibiotics in the culture medium would have an effect onthe nature of the protein biomarkers expressed, especially in thepre-translationally modified biomarker stage. Use of antibiotics isknown to suppress the expression of certain biomarkers, and wouldgenerally be expected to slow the growth of the bacteria in the sample.

The optimal conditions for short-term enrichment may vary, depending onthe organism. The method is applicable to both laboratory and clinicalbacterial samples.

As will be clear to those skilled in the art, diagnostic biomarkerprofiles for other bacterial strains are obtainable in a similar manneras has been described for MRSA. The mass spectrometric profile data thusgenerated can be used as reference data in the detection methods of theinvention. ‘Reference data’ therefore preferably comprises a massspectrometric profile, or information about particular peaks in the massspectrometric profile, which has been obtained for a short-term enrichedreference sample, known to contain a particular bacteria of interest. Areference sample may comprise a sample from a pure culture of thebacteria of interest, which may have been diluted to a suitableconcentration (i.e. comparable to the expected concentration of thebacteria at a screening site). The reference sample is added to aculture medium and subjected to short-term enrichment, as describedabove, to generate the reference biomarker profile.

The test described herein has the potential to detect the presence ofparticular bacteria at femtomole to picomole concentration of bacterialmolecules. The cost of the test compares very favourably with previousmethods, which can be very expensive and time-consuming. With the methodof the present invention, the time to diagnosis (from woundconcentration of bacteria to result) may be obtainable, for example,within two hours of the sample being taken.

A rapid diagnosis of a suspected bacterial infection or colonisationusing MALDI-TOF and short term enrichment is therefore obtainable usingthe methods of the invention. In general, diagnosis may involve thefollowing steps:

Step 1: A swab (or other sample) is taken from a patient. The type ofswab/sample may be varied depending on the type of organism to bedetected. However, it is envisaged that one uniform swab can bedeveloped so that, irrespective of what organism is being tested for,the same swab will still be suitable.

Step 2: The sample is inoculated into enrichment medium. The culturemedium and time period for enrichment can be chosen depending on thetype of bacteria. If a broth culture is used, the sample may becollected after culturing by a centrifugation/pooling process.

Step 3: The enriched sample is subjected to MALDI-TOF analysis. Aprofile is obtained and analysed by comparison with reference data forthe suspected bacterial infection. Different organisms will producedifferent profiles. Profiles may also differ slightly depending on theexact culture conditions used, although the general ‘early stage’profile will remain the same for each organism. The reference data towhich the obtained sample profile data is compared is chosenaccordingly.

Step 4: The result of the analysis is obtained from the MALDI-TOFanalysis—either positive (the suspected bacteria are present) ornegative (the suspected bacterial infection is not present).

It is envisaged that the majority of steps of this process can beautomated. For example, all of the steps after inoculation of the sampleinto the appropriate culture medium may be performed in a custom builtMS analysis machine, pre-programmed with the appropriate conditions(i.e. culture temperatures(s), time, centrifugation protocol) forshort-term enrichment of a number of bacteria, and with the referencedata for identifying the biomarker profiles of those bacteria.Alternatively, an automated system for performing the short-termenrichment may be provided, for use in conjunction with an existing massspectrometer.

The sample in the culture medium will be placed in the machine, and theappropriate program selected depending on the organism to be detected.The machine then heats the sample to the appropriate temperature forculturing, for example by immersing it in a water bath set at theappropriate temperature. After a pre-determined culture time (e.g. 30minutes to 2 hours), the machine will automatically perform thenecessary steps to prepare the sample for analysis. For example, thesample may be subjected to centrifugation, as described above, and theresulting solid sample transferred to a MALDI slide, within the machine.This may be done using a robotic system, for example.

This method shows the potential of mass spectrometry as a tool for rapiddiagnosis of bacterial infections/colonisations, such as MRSA.Preservation of the bacterial diagnostic profile in isolates aftershort-term enrichment is a significant development and raises thepossibility for the first time of wound swab to diagnosis within sixhours, which has the potential to revolutionize the isolation ofpatients and the endemicity of MRSA in hospitals and the community.

EXAMPLES

Materials:

The following ready prepared microbiological culture materials wereobtained from Oxoid, Basingstoke, UK: Nutrient Broth BO0210E andColumbia Agar with Horse Blood PB0122.

Brain Heart Infusion Broth CM0225 for the short term enrichment methodwas made as follows.

Reconstitution of the powder: The powder was commercially obtained fromOxoid. This was reconstituted by mixing 37 gm of the dry powder with onelitre of distilled water.

Autoclaving: The reconstituted solution was autoclaved at 121° C. forfifteen minutes. The solution was removed from the autoclave when thetemperature reached 80° C. (approximately fifteen to twenty minutes),then allowed to cool at room temperature for a further twenty to thirtyminutes.

Bottling: The autoclaved reconstituted powder was then bottled usingaseptic precautions into sterile glass containers.

This method of preparation showed consistently reproducible results.

Isolates Used:

-   -   EMRSA 15(Epidemic meticillin resistant Staphylococcus aureus        phage-type 15)-ten isolates,    -   EMRSA 16(Epidemic meticillin resistant Staphylococcus aureus        phage-type 16)-ten isolates,    -   MSSA (Meticillin sensitive Staphylococcus aureus)-ten isolates,    -   Coagulase negative Staphylococci (CNS)-ten isolates    -   Other Endemic MRSA's-ten isolates

Every isolate was tested in quintuplet and at concentrations of 10⁵colony forming units (cfu) as described in the methods section below.Profiles obtained after twenty four hours growth on a Columbia BloodAgar (CBA) were used as a comparative control.

Preparation of Matrix Solution:

The matrix is used to ionize the proteins within the sample which isneeded to help with separation of the molecules in the TOF tube. Theoptimal method of preparation of the matrix solution was published in2005 (Reference 14). The matrix used was5-chloro-2-mercaptobenzothiazole (CMBT) in a concentration of 3 mg/ml.

Example 1—Validation of the Method and Acquisition of Reference Data

The following steps were tested:

-   -   1. Short term enrichment.    -   2. Collection and cleaning of sample    -   3. MALDI-TOF processing.    -   4. Analysis

1. Short Term Enrichment:

Short term enrichment was carried out as described below:

The organisms were sub-cultured and grown overnight at 37° C. ColumbiaBlood Agar (CBA) to obtain a pure culture for further work. A singlecolony was inoculated into Nutrient Broth and incubated at 37° C.overnight to obtain an approximate concentration of bacteria of 10⁸colony forming units/millilitre. To determine the limits of detectionserial dilutions were carried out in Brain Heart Infusion (BHI) Broth,the broth used for the short term enrichment. This process isillustrated in FIG. 1. The serial dilutions result in samples withbacterial concentrations emulating those found at a wound site. Thesesamples were then incubated for a period of two hours in a water bath at37° C.

The short-term enriched samples are then prepared for MALDI-TOF analysisas follows:

2. Collection and Cleaning of Sample:

Step 1: After short term enrichment for two hours, all the sample ofeach isolate was divided into ten one milliliter aliquots, as shown inFIG. 2.

Step 2: All the aliquots were then centrifuged at 13500 rpm for threeminutes in a microcentrifuge.

Step 3: The supernatant was discarded and the pellet retained.

Step 4: 10 μl of water was added to the pellet and resuspended. All tenresuspended aliquots were pooled together. This was done for eachisolate.

Step 5: The pooled sample was then re-centrifuged at 13500 rpm for threeminutes.

Step 6: The resulting pellet from the pooled sample was then used forMALDI-TOF analysis.

3. MALDI-TOF Work:

All analysis was carried out after short term enrichment using theKratos Kompact MALDI 2 linear, bench-top instrument. A twenty wellKratos Kompact MALDI-TOF slide was cleaned and prepared using the methodoptimized by Jackson et al (Reference 14). This is shown in FIG. 3.

The method of preparation of the slide was as follows:

Step 1: The slide was layered with the bacterial isolates after shortterm enrichment at two hours, until all the pellet was used.

Step 2: The bacterial layered slide was then allowed to dry.

Step 3: This slide was then overlaid with matrix (CMBT) in two serialapplications of 0.5-1 μl each.

Step 4: The matrix layered slide was allowed to dry for 45 minutesbefore it was introduced into the MALDI-TOF for analysis

All work was repeated at two, four, six and twenty four hours.

Acquisition of data: The Kratos Kompact MALDI 2 is a linear, bench-topinstrument (FIG. 4). The instrument is equipped with a nitrogen laser(337 nm, 3 ns pulse width). The laser energy was adjusted to above thethreshold of ionization, enabling a balanced spectrum with peaksobserved in the 800-4000 Da mass range of interest. Ions wereaccelerated in the positive ion mode with an accelerating voltage of +20kV. The pulsed extraction of ions was optimized for 1000 Da. In allinstances five replicate spectra were obtained. For each replicate 100laser shots were accumulated by rastering across the width of the samplewell.

4. Data Analysis:

MALDI-TOF based analysis: The Intact cell MALDI spectrum was obtainedfrom 100 profiles acquired over the 2 μm spot on the target slide. Theseprofiles were compiled automatically by the instrument to form anaverage profile for the replicate. Five replicates were obtained foreach isolate, representing data compiled from 500 individual profiles.Once these replicates were obtained using the analysis tools within theMALDI-TOF instrument the following was carried out:

a) Determining the ‘best replicates’ using cluster analysis:

Using prototype analytical tools, the 5 replicates were subjected tocluster analysis using a modified Jaquard algorithm designed toaccommodate the large data set (over 16000 data points) and to take intoconsideration the operational issues associated with theinstrumentation. Using this algorithm with the following parameters (cutoff parameters; threshold of 0.5 mV, a mass range of 500 daltons to10000 daltons and a threshold ratio of 4 (only those peaks with abaseline to peak ratio of greater than 4 were included)) the replicateswere clustered and those with a sequence dissimilarity of greater thanforty percent were discarded from the next step of analysis (FIG. 5).

b) Combining files:

Only those replicates with sequence dissimilarity of less than fortypercent were included in this element of the analysis. These replicateswere then used to create a combined file using a preset programme withinthe Kratos Kompact MALDI-TOF software. The data from these combinedfiles was used for all further analysis.

This pattern was maintained for each isolate of every species and forall the data obtained from two, four, six and twenty-four hour work. Thebroths which were used for the control experiments were also subjectedto the same data analysis.

Manual Analysis: Once the combined files were obtained for each of theisolates the following was carried out:

a) Formulation of mass lists:

Each combined file consisted of peak profiles that were unique to eachisolate. Each profile contained within it a list of the peak masses.This is the mass list. The peak list of the combined file of an isolateis shown below.

shtermmar07dhr CMBT E15FSCDHR Data: E15FSCdhr0001_ic_comb_. 16 13 Mar.2007 16:08 Factory calibrated Kratos PCKompact SEQ V1.2.0: + LinearHigh, Power: 80, P.Ext. @ 1046 (bin 57) Mass % Total Apex (mV) 1017.590.39 1.56 1026.07 0.18 0.83 1034.28 0.12 0.81 1041.30 0.54 2.26 1046.190.11 0.79 1057.25 0.17 0.84 1095.17 1.32 6.06 1111.53 0.09 0.81 1116.284.92 22.69 1132.48 3.06 13.93 1140.14 4.07 16.16 1155.87 3.00 9.661162.00 0.18 1.21 1167.17 0.06 0.64 1170.08 0.12 0.78 1178.20 0.36 1.851194.19 0.05 0.63 1216.55 0.11 0.84 1229.14 0.08 0.66 1252.50 0.09 0.59

All peaks in the mass range of eight hundred daltons to five thousanddaltons were noted for each of the ten isolates for each species ofStaphylococci. This data was then transferred to a spreadsheet as shownin Table 1 below, which is an example of a mass list created from thecombined files for the ten isolates of S. aureus.

TABLE 1 SA1 SA2 SA3 SA4 SA5 SA6 SA7 SA8 SA9 SA10 1017.29 1008.24 1017.861001.93 1001.93 1008.84 1017.59 1001.93 1018.47 1010.05 1023.94 1017.291024.86 1009.14 1004.33 1018.49 1024.85 1010.05 1025.47 1016.98 1026.071024.25 1034.33 1019.4 1010.05 1025.16 1033.97 1018.19 1034.33 1020.311033.67 1033.67 1041.39 1024.85 1017.59 1034.28 1040.99 1025.46 1041.71024.55 1040.07 1040.99 1095.55 1033.97 1024.25 1040.99 1057.56 1034.281057.74 1033.67 1043.44 1056.95 1117.08 1040.99 1033.97 1057.56 1062.811040.99 1095.86 1040.38 1050.18 1063.11 1133.37 1057.87 1040.69 1078.931071.16 1057.56 1112.31 1047.11 1056.64 1067.75 1140.76 1062.5 1045.891095.17 1078.93 1062.5 1117.4 1051.1 1078.3 1071.47 1156.57 1064.351050.18 1111.53 1090.16 1078.93 1133.37 1057.56 1094.23 1078.93 1163.061068.37 1056.95 1116.59 1095.48 1095.48 1141.08 1061.88 1104.59 1083.291171.19 1073.33 1062.5 1132.48 1100.19 1099.25 1157.22 1068.99 1110.591087.66 1179.35 1078.62 1068.06 1140.14 1111.22 1105.22 1163.38 1071.471115.64 1094.86 1217.26 1084.22 1070.85 1156.19 1116.59 1111.53 1171.521078.62

b) Use of macros:

Once the mass lists were created for each of the five species ofStaphylococci a specially designed macro within the spreadsheet was usedto look for common biomarker peaks. This macro identified within acorrect range peaks with 0.2% accuracy.

The common peaks could be highlighted as shown in Table 2 below. Thisaided visual identification.

TABLE 2 SA1 SA2 SA3 SA4 SA5 SA6 SA7 SA8 SA9 SA10 1001.93 1001.93 1001.931004.33 1008.24 1009.14 1010.05 1008.84 1010.05 1010.05 1017.29 1017.291017.86 1017.59 1018.49 1017.59 1018.19 1018.47 1016.98 1019.4 1020.311023.94 1024.25 1024.86 1024.85 1024.25 1025.16 1024.85 1025.46 1025.471024.55 1026.07 1033.67 1033.67 1034.33 1033.97 1033.97 1034.28 1033.971034.28 1034.33 1033.67 1040.07 1040.99 1041.39 1040.99 1040.69 1040.991040.99 1040.99 1041.7 1040.38 1043.44

For further reproducibility each of the lists were visually verified andany peaks that were not highlighted but were within five daltons of thelowest peak in the same mass range were added to the list of commonpeaks. Thus for each of the five species a list of peaks was drawn upwhich was common to all ten isolates of the same species.

c) Derivation of final diagnostic profiles:

The final diagnostic profile which identified the peaks common to allthe Staphylococci as well as those unique to MRSAs was drawn up bysubjecting the common lists of each of the species to scrutiny by thesame Excel macros spreadsheet.

This detailed analysis has been carried for all the bacteria at two,four, six and twenty four hours and diagnostic profiles have beenobtained at each of these time points.

Results:

-   -   1) For the first time a provisional diagnostic profile for MRSA        is proposed on the basis of proteomic analysis of laboratory        based isolates after short term enrichment (FIG. 6).    -   2) The provisional diagnostic profile of MRSA is maintained in        the bacteria in the mass range of 1-4 kDa with 100%        reproducibility across the spectrum of short term enrichment        (FIG. 7).    -   3) A provisional diagnostic profile for MRSA at two hours:

Peaks that are common in all the isolates of Staphylococci:

1018.19, 1034.28, 1041.6, 1057.56, 1095.8, 1116.91, 1133.11, 1140.46,1156.19, 1170.73, 1216.55, 1315.64, 1353.01, 1379.63, 1437.58, 1453.79,1520.28, 1536.2, 1552.21, 1558.19, 1574.31, 1633.88, 1665.82, 1682.1,1952.5, 1968.44, 1990.35, 2029.85, 2051.25, 2100.46, 2116.12, 2181.99,2215.31, 2237.21, 2524.61, 2540.82, 2578.69, 2600.86, 2623.62, 2639.66,2661.6, 2677.76

Peaks that, are unique to the isolates of MRSA: 1080.17, 1179.5,1651.16, 1866.8, 1882.39, 1931.2, 2068.02, 2074.49, 2085.72, 2139.28,2155.08, 2160.8, 2200.17, 2254.27, 2400.97, 2580.13, 2875.41

-   -   4) The peak profiles at short term are significantly different        to those obtained at twenty four hours in agar plate growth (see        FIG. 8).    -   5) The test has the potential to detect at femtomole to picomole        concentration of molecular molecules from bacteria    -   6) Cost of the test is approximately 5 p/sample.    -   7) Time to diagnosis from wound concentration of bacteria to        result can be obtained as early as two hours.

This test shows the potential of mass spectrometry as a tool for rapiddiagnosis of MRSA.

Example 2—Validation of Results with Clinical Samples

1. Clinical swabs are taken—screening sites include nose, groin, axillaand perineum, as well as swabs from ulcers, blood cultures and tissues.

2. A portion of each sample is inoculated into custom BHI broth,prepared as in Example 1. For comparison, each sample is also culturedaccording to the ‘gold standard’ for MRSA detection i.e. culturing for16-72 hours on selective or non-selective Agar plates.

3. The BHI broth samples is introduced into the automated prototype.Culturing is carried out for approximately 2 hours, by incubation in awater bath. Samples are prepared for analysis, as described in Example1.

4. MALDI-TOF analysis is carried out to determine the MS profile of thesamples. These are compared (e.g. by a data processor within theprototype) with the reference data (e.g. as shown in Example 1), todetermine the presence or absence of MRSA.

5. Results are compared with the long-term culture ‘gold standard’methods, using the following criteria: specificity, sensitivity,accuracy, false-positive rate, false-negative rate and cost economics.

REFERENCES

1) Ashimoto, A., T. Hamada, et al. (1995). “Molecular epidemiology ofStaphylococcus spp. contamination in the ward environment: study on mecAand femA genes in methicillin-resistant strains.” Kansenshogaku Zasshi69(1): 15-20.

2) Brakstad, O. G. and J. A. Maeland (1995). “Direct identification ofStaphylococcus aureus in blood cultures by detection of the geneencoding the thermostable nuclease or the gene product.” Apmis 103(3):209-18.

3) Francois, P., D. Pittet, et al. (2003). “Rapid detection ofmethicillin-resistant Staphylococcus aureus directly from sterile ornonsterile clinical samples by a new molecular assay.” J Clin Microbiol41(1): 254-60.

4) Zhang, K., J. Sparling, et al. (2004). “New quadriplex PCR assay fordetection of methicillin and mupirocin resistance and simultaneousdiscrimination of Staphylococcus aureus from coagulase-negativestaphylococci.” J Clin Microbiol 42(11): 4947-55.

5) Hardy, K. J., A. Szczepura, et al. (2007). “A study of the efficacyand cost-effectiveness of MRSA screening and monitoring on surgicalwards using a new, rapid molecular test (EMMS).” BMC Health Sery Res 7:160.

6) Jeyaratnam, D., C. J. Whitty, et al. (2008). “Impact of rapidscreening tests on acquisition of meticillin resistant Staphylococcusaureus: cluster randomised crossover trial.” Bmj 336(7650): 927-30.

7) Cunningham, R., P. Jenks, et al. (2007). “Effect on MRSA transmissionof rapid PCR testing of patients admitted to critical care.” J HospInfect 65(1): 24-8.

8) de San, N., O. Denis, et al. (2007). “Controlled evaluation of theIDI-MRSA assay for detection of colonization by methicillin-resistantStaphylococcus aureus in diverse mucocutaneous specimens.” J ClinMicrobiol 45(4): 1098-101.

9) Chediac-Tannoury, R. and G. F. Araj (2003). “Rapid MRSA detection bya latex kit.” Clin Lab Sci 16(4): 198-202.

10) Edwards-Jones V, Claydon, M. A., Evason, D. J., Walker, J., Fox, A.J., and Gordon, D. B. (2000). “Rapid discrimination betweenmethicillin-sensitive and methicillin-resistant Staphylococcus aureus byintact cell mass spectrometry” J. Med. Microbiol. 49, 295-300.

11) Jackson, K. A., Fox, A. J., Edwards-Jones, V. (2003) “Determinationand structural examination of potential biomarkers forMethicillin-resistant Staphylococcus aureus.” Applications of genomicsand proteomics for analysis of bacterial biological warfare agents—IOSPress—Editors: DelVecchio, V. G., Krcmery, V

12) Smole S C, King L A, Leopold P E, Arbeit R D. “Sample preparation ofGram-positive bacteria for identification by matrix assisted laserdesorplion/ionization time-of-flight.” J Microbiol Methods 2002; 48:107-15.

13) Bright J J, Claydon M A, Soufian M, Gordon D B. “Rapid typing ofbacteria using matrix-assisted laser desorption ionixationtime-of-flight mass spectrometry and pattern recognition software.” JMicrobiol Methods 2002; 48: 127-38.

14) Jackson, K. A., V. Edwards-Jones, et al. “Optimisation of intactcell MALDI method for fingerprinting of methicillin-resistantStaphylococcus aureus.” J Microbiol Methods 2005, 62(3): 273-84.

15) UK Patent Publication GB 2 3438 066 A-Bruker Daltonik GmbH (14 Nov.2007).

16) Bendy R H, Nuccio P A, Wolfe E, Collins B, Tamburro C, Glass W,Martin C M. “Relationship of quantitative wound bacterial counts tohealing of decubiti. Effect of topical gentamicin.” Antimicrob AgentsChemother. 1964;4:147-155.

17) Hanft, Jason R. and Smith, Brigette, “How To Differentiate BetweenInfected Wounds And Colonized Wounds”, Podiatry Today, 2005, 18, 7,85-90.

1. A method for detection of the presence or absence of particularbacteria in a clinical sample, the method comprising: (i) enriching theclinical sample by culturing the bacteria present in the sample for aperiod of time less than 6 hours such that pre-modification biomarkersare produced in the sample, wherein enrichment is carried out by:inoculating the bacterial-containing clinical sample into a broth;culturing the clinical sample-containing broth for a period of less than6 hours; and separating an enriched clinical sample from thesample-containing broth by centrifugation, and wherein said broth isprepared by a method comprising: reconstituting a powderedmicrobiological culture material in water; autoclaving the reconstitutedsolution at a temperature between 80 and 150° C.; leaving the solutionto cool in the autoclave to a pre-determined temperature of between 60and 90° C.; removing the solution from the autoclave when thepredetermined temperature is reached; and allowing the solution tocontinue cooling at room temperature; (ii) analysing the enrichedclinical sample by mass spectrometry, to obtain biomarker profile datafor the clinical sample, wherein the biomarker profile data comprisespeaks of specific masses corresponding to pre-modification biomarkersproduced by the bacteria in the sample; (iii) correlating the clinicalsample biomarker profile data with reference data to look for peakshaving specific masses common to the sample biomarker profile data andto the reference data, to determine the presence of absence of saidparticular bacteria in the sample; wherein a match between the masses ofthe peaks of the biomarker profile of the enriched clinical sample andthe masses of the peaks of the biomarker profile of the referenceclinical sample indicates the presence of the particular bacteria in thesample, wherein the reference data relates to pre-modificationbiomarkers represented by peaks having specific masses produced by areference sample of said particular bacteria which has been cultured forless than six hours, and wherein the concentration of bacteria in theclinical sample before enrichment is about 10⁷ cfu/ml or below.
 2. Amethod according to claim 1, wherein the pre-modification biomarkers arepre-modification proteins, peptidoglycans, glycoproteins, glycopeptides,carbohydrates, and/or lipopolysaccharides.
 3. A method according toclaim 2, wherein the pre-modification proteins are pre-PTM(posttranslational modification) proteins.
 4. A method according toclaim 2, wherein the pre-modification biomarkers comprise unglycatedcell wall peptides.
 5. A method according to claim 1, wherein thepre-modification biomarkers comprise biomarkers with a molecular massbelow 4000 Da, or from 1000 to 4000 Da.
 6. A method according to claim1, wherein the culturing in step (i) is carried out for four hours orless.
 7. A method according to claim 1, wherein the culturing in step(i) is carried out for two hours or less.
 8. A method according to claim1, wherein the said bacteria is a Gram positive bacteria.
 9. A methodaccording to claim 1, wherein said bacteria is a Staphylococcus.
 10. Amethod according to claim 9, wherein said bacteria ismethicillin-resistant Staphylococcus aureus.
 11. A method according toclaim 1, wherein the sample is a clinical sample taken from a humanpatient.
 12. A method according to claim 1, wherein the sample is takenfrom a wound site, an ulcer, or a screening site.
 13. A method accordingto claim 1, wherein said biomarker profile data is proteomic profiledata.
 14. A method according to claim 1, wherein the analysis by massspectrometry is performed using MALDI-TOF mass spectrometry.
 15. Amethod according to claim 1, wherein the broth is a Brain Heart InfusionBroth, a Mueller-Hinton Broth, an anaerobic broth or a nutrient broth.16. A method according to claim 1, wherein culturing the samplecomprises incubation in a water bath.
 17. A method according to claim 1,wherein the centrifugation step comprises: dividing thesample-containing broth into a plurality of aliquots; centrifuging thealiquots; discarding the resultant supernatants; re-suspending thepellets in water; pooling the resultant pellets; and centrifuging asuspension of the pooled pellets, to separate the pellet containing theenriched sample.
 18. Apparatus for use in a method according to claim17, comprising a mass spectrometer and a data processor, the processorbeing programmed with said reference data for identifying one or moreparticular bacteria, the reference data comprising peaks of thebiomarker profiles of each particular bacteria, wherein the biomarkerprofiles of the particular bacteria have been obtained by enriching asample of each particular bacteria by culturing for a period of timeless than 6 hours.
 19. Apparatus according to claim 18, wherein the massspectrometer is a MALDI-TOF mass spectrometer.
 20. Apparatus accordingto claim 18, comprising an automated system for culturing a sample foranalysis in the mass spectrometer.
 21. Apparatus according to claim 20,wherein the automated system comprises: an incubator for heating thesample during culturing; a temperature control device for controllingthe temperature of the incubator; a centrifuge for separation of thecultured sample from the culture medium.
 22. The method of claim 1,wherein the particular bacteria being detected is selected from thegroup consisting of Staphylococci, Clostridium, Escherichia coli,Campylobacter, Salmonella, Pseudomonas, Shigella, Neisseria, Klebsiella,Vibrio, Legionella, H. Influenza, H. pylori, Bacillus, Listeria, andbacteria causing tuberculosis and leprosy.